青钱柳（Cyclocarya paliurus）属于胡桃科青钱柳属植物，是集药用、材用、保健等于一体的多功能树种，具有典型的雌雄异型异熟特征（雌先型：protogyny, PG；雄先型：protandry，PA）。此外，青钱柳在自然界中具有二倍体和四倍体两种倍性，且以四倍体植株占绝对优势，其基因组基础、雌雄异熟的分子机制、三萜类代谢过程仍然知之甚少。近日，南京林业大学方升佐/洑香香教授联合中国农业科学研究员深圳农业基因组所张兴坦研究员在Genomics, Proteomics & Bioinformatics发表题为“Whole-genome Duplication Reshaped Adaptive Evolution in A Relict Plant Species, Cyclocarya paliurus”的研究论文，为基因组辅助青钱柳人工林资源培育提供了宝贵的基因组资源。该研究组装三个高质量青钱柳基因组：2个二倍体和1个同源四倍体；随后在全基因组复制（WGDs）事件分析中发现，近期的一次WGD事件导致了该物种的同源四倍化，并且四倍体表现出更强的光合作用和三萜类代谢能力；鉴定到P450家族中的剂量效应基因，其可能在青钱柳酸B的生物合成中发挥了关键作用；筛选到了与青钱柳雌雄异熟开花习性相关的关键基因，特别是赤霉素通路相关基因。45份青钱柳材料重测序分析结果，发现大量受选择基因参与了植物生长发育和三萜类代谢，在群体进化历史中发现了两次大的瓶颈期且与两次大环境变化事件相吻合。本研究为研究青钱柳的基因组进化、雌雄异熟特征、三萜类代谢过程和进化历史提供了深刻的见解，并为其重要性状特征的定向培育提供基因组学资源。

1. 青钱柳染色体水平的基因组组装和基因注释

4.通过对青钱柳的基因组测序，使用PacBio、HiC和Illumina测序数据，研究人员组装了PA-dip、PG-dip和PA-tetra三个基因组，组装大小分别为586.62 Mb、583.45 Mb和2.38 Gb。其中PA-dip、PG-dip和PA-tetra的Contig N50分别达到1.9 Mb、1.4 Mb和431 Kb，BUSCO完整度分别为95.2%、96.4%和95.5%。三个基因组分别注释出34,699、35,221和90,752个蛋白质编码基因，其中PA-tetra中共34,633个编码蛋白质的等位基因。另外，三个基因组中分别鉴定出282.25 Mb（组装基因组大小的48.1%）、316.95 Mb（54.3%）和1,154.35 Mb（48.4%）重复序列，PG-dip基因组重复序列比例稍大于其他两个基因组。

Figure 1 Morphology and genome duplications of Cyclocarya paliurus

A. Leaves. B. Mature fruits. C. Female and male flowers of PA-dip. D. Female and male flowers of PG-dip. E. Fruit wings that start to spread. F. Tetraploid plant. G. Genomic alignments between the basal angiosperm Amborella trichopoda, and the basal eudicot Vitis vinifera, as well as PG-dip, PA-dip, and PA-tetra C. paliurus are shown. The conserved collinear blocks are shown as the gray lines in the background and the green lines indicate cases in each round of WGD. WGD, whole-genome duplication; PA-tetra, the protandrous tetraploid C. paliurus; Chr, chromosome; PG-dip, the protogynous diploid C. paliurus; PA-dip, the protandrous diploid C. paliurus.

2. 青钱柳同源四倍体基因组中的独立WGD事件

为了研究青钱柳系统进化位置，对包括青钱柳在内的9个物种进行系统进化分析。结果显示青钱柳与同科的枫杨属植物互为姐妹群，分化时间约在46.07百万年前，与化石记录时间相近。此外，相对于青钱柳和枫杨的共同祖先，青钱柳基因组中有285个基因家族发生了扩张，264个基因家族发生了收缩；扩张的基因家族主要参与青钱柳三萜类化合物的生物合成。每个同源基因对的同义替换率（Ks）分布呈现3个峰，即代表了3个全基因组加倍（WGD）事件。除了与葡萄共同经历的WGT（γ）事件，青钱柳在近期经历了两次独立的全基因组WGD事件。PA-dip和PG-dip间的共线性分析验证了早期WGD1的准确性，可追溯到~67.6-50.7 Mya；最近一次WGD2事件发生在~11.2-10.5 Mya，由此产生了青钱柳四倍体植株。

Figure 2 Phylogenetic and comparative analysis of C. paliurus

A. Phylogenetic relationship of C. paliurus, Carya illinoinensis, Juglans nigra, Pterocarya stenoptera, Arabidopsis thaliana, Ziziphus jujuba, V. vinifera, Populus trichocarpa, and Oryza sativa. The divergence times among different plant species are labeled in the bottom. B. Venn diagram of orthologous and species-specific genes in different plant genomes. C. Evolutionary analysis of the diploid and tetraploid C. paliurus genomes with the distribution of Ks of orthologs. D. Synteny analysis between PA-tetra and PA-dip genomes. The full name “monoploid” indicates a reference genome assembly with only one representative haplotype retained, while the “haplotype” indicates fully phased genome with all the four haplotypes. Ks, synonymous substitution rates; MYA, million years ago.

3. 基因剂量效应对青钱柳光合作用及叶内三萜类化合物积累的影响

幼苗形态、解剖结构和叶片光合能力研究表明，相比于二倍体，四倍体光合能力较强，植株生长更快。为了研究多倍化对植物功能的影响，研究发现691个基因在四倍体中表达明显高于二倍体（fold change ≥ 2 and P value ≤ 0.05），这些基因被定义为剂量效应基因。此外，GO和KEGG注释显示这些基因在一些主要的生物学通路中大量富集。其中，涉及“carbohydrate”、 “starch”、“sucrose”、“alanine”、“aspartate”、“glutamate metabolism”、“phosphatidylinositol signaling system”和“ion channels”等等，并在光合作用中起着至关重要的作用，是植物生长发育和胁迫反应中不可或缺的部分。同时，我们也鉴定出759个剂量补偿效应基因，在二倍体中表达量高于四倍体（fold change ≥ 2 and P value ≤ 0.05）。富集结果显示，大量的剂量补偿效应基因在“regulation of DNA recombination”、“maltose metabolic process”、“cysteine and methionine metabolism”、“amino sugar”、“nucleotide sugar metabolism pathways” 等显著富集。

Figure 3 Dosage-effect contributes to increased growth adaptability and accumulation of terpenoids.

A. Scanning electron microscopy of stomata in diploid and tetraploid C. paliurus leaves. SL, stomatal length; SA: stomatal aperture; SW: stomatal width; mag, magnification; WD, working distance; HV, high voltage; AM, ante meridiem; PM, post meridiem. B. The comparison of chlorophyll content between diploid and tetraploid C. paliurus. Statistical significance (n = 5) was determined using the two-sided Student’s t test. Error bars indicate mean ± SD of indicated replicates. **, P value < 0.01. C. The comparison of Pn between diploid and tetraploid C. paliurus. ***, P value < 0.001; SD, standard deviation; Pn, net photosynthetic rate. D. Heatmap showing the accumulation patterns of triterpenoids among the four samples (Sept_diploid, diploid samples collected in September; Sept_tetraploid, tetraploid samples collected in September; May_diploid, diploid samples collected in May; May_tetraploid, tetraploid samples collected in May). E. Expression profiles of genes associated with CA-B synthesis in C. paliurus tender and mature leaves for different ploidy. FPP, farnesyl pyrophosphate; SQS, squalene synthase; SQE, squalene epoxidase; bAS, β-amyrin synthesis. The scale ranging from blue (low) to red (high) indicates the expression magnitude of the FPKM values. The genes of CYP72A subfamily are shown in blue. FPKM, fragments per kilobase per million; CA-B, cyclocaric acid B.

4. 青钱柳雌雄异熟特征相关基因的挖掘

青钱柳具有典型雌雄异型异熟特征，即PA或PG个体中功能分离，在个体水平上，雌、雄花期不遇；在群体水平上，不同交配类型间的雌雄花期则表现出同步性。这种开花机制可避免自交，促进远缘杂交；但也可能导致种子饱满度低，影响其资源扩繁。为了鉴定调控青钱柳雌雄异熟的相关基因，根据开花物候，将花芽发育过程划分为五个发育时期，对各阶段的转录组进行测序与分析。通过比较转录组、共表达网络以及花芽发育过程中内源赤霉素含量的检测，挖掘调控青钱柳花芽发育的分子机理。

对PG和PA雌花芽（PG- F vs. PA- F）进行两两比较，鉴定到958个差异表达基因（DEGs）。同样，在PA和PG的雄花芽（PA-Mvs.PG- M）中鉴定2373个DEGs。富集结果表明这些DEGs在花器官形成和发育的一系列生物过程中显著富集。值得注意的是，大量上调的DEGs （PG-F中的128/855和PA-M中的58/1539）与激素的生物合成和信号通路有关。随后检测了PA和PG花芽中内源激素含量，包括赤霉素(GA₃)、生长素(IAA)和脱落酸(ABA)，发现GA₃含量在交配类型间（PG-F_vs_PA-F和PA-M_vs_PG-M）存在显著差异。研究认为，GA₃含量可能在调节花芽生理分化中起着至关重要的作用，并与雌雄异熟紧密关联。

Figure 4 Identification and functional enrichment of DEGs between PG and PA C. paliurus at five flowering time stages (S0–S4).

Venn diagrams of DEGs in the female floral buds (PG-F vs. PA-F). B. Venn diagrams of DEGs in the male floral buds (PA-M vs. PG-M). C. GO enrichment of the 958 DEGs in female floral buds (PG-F vs. PA-F). D. GO enrichment of the 2373 DEGs in male floral buds (PA-M vs. PG-M). In all subsequent figures, PG-F means female floral buds of PG, PA-F means female floral buds of PA, PG-M means male floral buds of PG, and PA-M means male floral buds of PA. FC, fold change; DEGs, differentially expressed genes; PG, protogyny; PA, protandry.

基于22份RNA-seq数据，进行共表达网络分析，共保留了20829个基因，分布在26个模块中。我们观察到三个模块中的基因（darkorange: 47 genes; pink: 912 genes; red: 1244 genes）与GA₃含量高度相关（R² ≥ 0.56, P value ≤ 0.007, Pearson test），这些基因除了参与激素和赤霉素的合成与代谢，还在“signal transduction”、 “response to stimulus and stress”、“biological regulation” 等方面发挥重要作用。此外，研究人员还鉴定到关键hub转录因子（TF），如Trihelix-1 (CpaF1st06806)、ERF066 (CpaF1st15865)、ERF090 (CpaF1st01445)和WRKY55 (CpaF1st00113)等在内源激素信号传递和花发育、胁迫信号转导、植物激素介导中发挥重要作用。

Figure 5 Functions and networks of co-expression module genes

A. Module–trait relationships. The column indicate GA₃, and the rows indicate the different modules. The red and blue colors indicate positive and negative correlations, respectively. The correlation coefficient (r) and P value (P) are displayed in each cell. B. Eigengene expression profiles in the darkorange module. C. Eigengene expression profiles in the pink module. D. Eigengene expression profiles in the red module. E. Construction of the correlation network of the pink module. F. Construction of the correlation network of the red module. The large circles represent transcription factors. The color is determined by the edge number of the gene.

5. 群体遗传学揭示青钱柳的进化历史

对45份来自9个不同地理居群的青钱柳材料进行了系统发育关系分析，所有样本分为两个组。10个二倍体样本聚集在第一组并且与外类群亲缘关系较近。剩余35个四倍体样本分在第二组。主成分（PCA）和群体结构分析均支持这种群体结构，表明在青钱柳物种中，最后一次WGD事件的起源是单一的，而不是在其他多倍体物种中观察到的多个起源。研究人员对二倍体和四倍体分别进行了选择性清除分析，二倍体和四倍体基因组中分别有1528和4812个基因受到选择，这些基因主要参与次生代谢、DNA重组和麦芽糖代谢过程等。此外，四倍体特有的受选择基因大多富集到萜烯生物合成途径，这可能是由于四倍体具有较强的环境适应性和胁迫耐受性。研究人员通过群体历史动态分析发现了两次大的瓶颈期并且和两次大环境变化事件相吻合。

Figure 6 Phylogenetic splits among C. paliurus populations.

A. Dispersion of 45 individuals sampled from 9 sites across most of the geographic range of C. paliurus. Populations are plotted with dots color-coded based on dispersion by latitude and longitude. Yellow color stars represent the co-presence of diploid and auto-tetraploid distribution, blue color stars represent only auto-tetraploid distribution, and purple color star represents the outgroup (Juglans regia) (more details were shown in Table S14). The world map was constructed from the Natural Earth dataset (http://www.naturalearthdata.com). B. A phylogeny for C. paliurus accessions, estimated from SNPs in neutrally evolving sites. C. PCA shows clear separation between diploid and auto-tetraploid populations. D. ADMIXTURE plot for C. paliurus showing the distribution of K = 2, 3, 4, and 5 genetic clusters, among them, K = 2 (representing the divergence within diploid and auto-tetraploid clades) genetic clusters indicates the smallest CV error. CV plots display CV error versus K, which suggests that K = 2 is also the best fit. SNPs, single nucleotide polymorphisms; PCA, principal component analysis; CV, cross-validation.

      该研究由多家单位研究人员共同协作完成。南京林业大学林学院瞿印权博士（现任职浙江海洋大学）和尚旭岚副教授为论文的共同第一作者。南京林业大学林学院方升佐教授、洑香香教授、中国农科院深圳农业基因组所张兴坦研究员为论文的共同通讯作者。

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